Abstract 1490: Dual chlorotoxin and methylguanine methyltransferase γδ-T cells for drug resistant immunotherapy of glioblastoma multiforme

Abstract

Abstract While recent advances in immunotherapies have shown promise in extracranial tumors, Glioblastoma Multiforme (GBM) has remained impervious to these advances with a consistent median survival of 15 months. We have developed a novel approach to the treatment of primary GBM by combining simultaneous intracranial administration of gene-modified γδ T cells and standard temozolomide (TMZ) maintenance chemotherapy. These γδ T cells are transduced with O-6-Methylguanine-DNA Methyltransferase (MGMT), conveying resistance to alkylating chemotherapies, thereby allowing effector function at therapeutic concentrations of TMZ when tumor NKG2DL expression is significantly elevated. We modified γδ T cells with a lentivector construct expressing a chimeric antigen receptor (CAR) with a chlorotoxin binding domain (CLTX-CAR) to improve GBM targeting. CLTX is a small natural peptide derived from scorpion venom that specifically targets altered expression of matrix metalloproteinase 2 (MMP2) and chloride channel CLCN3 on GBM. MGMTp140k was co-expressed within the same CLTX-CAR vector to confer TMZ resistance to the CAR-T cells. The CLTX-CAR vectors contain a CD8α signal peptide, a mono or dual CLTX binding domain, a Myc-Tag peptide, a CD8α hinge domain, a CD28 transmembrane domain, and a costimulatory domain followed by a CD3ζ activation domain. We used P2A peptide to co-express MGMTp140k with the CAR. Initially, we demonstrated efficient transduction of the CLTX-CAR with a dual-CLTX construct as the binding domain (dCLTX-CAR) in Jurkat T cells. Cell surface localization of the dCLTX-CAR was verified by flow cytometry. When compared with the mono-CLTX-CARs (mCLTX-CARs) transduced Jurkat cells, the dCLTX-CARs demonstrated increased CD69 activation (MFI= 4873 vs 1078) and greater cytotoxicity against tumor cells in vitro. The modified T cells also demonstrated enhanced cytotoxicity against tumor cells under TMZ exposure in vitro. Since γδ T cells recognize and kill tumors through NKG2DL stress antigen recognition, we hypothesized that a CLTX-CAR without an activation signal may be sufficient to enhance recognition and cytotoxicity of γδ T cells to tumor cells and mitigate CAR-T activation-induced cell death. We then developed dCLTX-CAR constructs without the CD3ζ domain (dCLTX- Δz-CARs). As expected, the dCLTX-Δz-CAR lentivirus transduced Jurkat cells demonstrated enhanced cell-cell binding compared to the full dCLTX-CAR but no CD69 expression when co-cultured with GBM cells. Overall, we were able to generate dCLTX-CAR T cells with resistance to TMZ and showed improved activation and cytotoxicity against GBM cells under TMZ exposure. Our approach of combining the dCLTX-CAR and TMZ resistance will be further validated in animal model experiments and could be a potential candidate for clinical development for GBM. Citation Format: Lei Ding, Lawrence S. Lamb. Dual chlorotoxin and methylguanine methyltransferase γδ-T cells for drug resistant immunotherapy of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1490.